Ascorbate is the most abundant soluble antioxidant in plants and is also an essential nutrient for humans and a few other animals. Ascorbate contributes significantly to the overall intake of “free radical scavengers” or “anti-oxidative metabolites” in the human diet. Convincing evidence now shows that such metabolites either singly or in combination, benefit health and well-being, acting as anti-cancer forming agents and protecting against coronary heart disease.
Almost all of the dietary ascorbate intake in humans is derived from plant products. The ascorbate content of plant tissues however, is remarkably variable. Whilst leaf ascorbate content is generally high and relatively uniform in herbaceous and woody plants, a huge and unexplained variability in ascorbate content found is in non-green edible plant tissues. For example, in fruits, the levels vary from up to 30 mg gFW-1 AsA in the camu camu of Mirciaria dubia, to less than 3 μg gFW-1 AsA in the medlar of Mespilus germanica (Rodriguez et al. 1992, J Chromatogr Sci, 30:433-437). A range of values for ascorbate have been reported in kiwifruit (Ferguson, A. R., Botanical nomenclature: Actinidia chinensis, Actinidia deliciosa, and Actinidia setosa. Kiwifruit: science and management, ed. I. J. Warrington and G. C. Weston. 1990, Palmerston North; New Zealand: New Zealand Society for Horticultural Science. 576. Beever, D. J. and G. Hopkirk, Fruit development and fruit physiology. Kiwifruit: science and management, ed. I. J. Warrington and G. C. Weston. 1990, Palmerston North; New Zealand: New Zealand Society for Horticultural Science. 576.) Ascorbate content of fruits from different vines range for A. deliciosa, 30-400 mg/100 g (Ferguson, A. R., 1991 Acta Hort. 290: p. 603-656, Spano, D., et al., 1997 Acta Hort., 444: p. 501-506.) while for the cultivar ‘Hayward’ the reported range is 80-120 mg/100 g (Beever, D. J. and G. Hopkirk, Fruit development and fruit physiology. Kiwifruit: science and management, ed. I. J. Warrington and G. C. Weston. 1990, Palmerston North; New Zealand: New Zealand Society for Horticultural Science. 576). Higher concentrations of ascorbate are reported in fruit of, A. arguta, A. chinensis (Muggleston, S., et al., Orchardist, 1998. 71(8): p. 38-40, Chen, Q. and Q. Chen, Crop Genetic Resources, 1998(2): p. 3, Coggiatti, S., 1971 Ital Agr, October, 108(10): p. 935-941) A. chrysantha and A. polygama with very high levels in A. eriantha, and A. latifolia (>1% fresh weight) (Ferguson 1991 Acta Hort. 290: p. 603-656. and A. kolomikta (Kola, J. and J. Pavelka, 1988 Nahrung, 32(5): p. 513-515).
Three pathways of biosynthesis of ascorbic acid have been proposed in plants, one through L-Gal (Wheeler et al., 1998, Nature 393, 365-369), another from myoinositol (Loewus & Kelly, 1961, Arch. Biochem. Biophys. 95, 483-493; Lorence et al., (2004) Plant Physiol. 134, 1200-1205) and a third through Galacturonic acid (Agius et al., 2003, Nat Biotechnol 21, 177-81). The L-Gal pathway proceeds through L-Gal to galactono-1,4-lactone and thence to ascorbate (Wheeler et aL, 1998, Nature 393, 365-369).
To date, all the genes encoding enzymes, and their associated enzymatic activities, for the L-Galactose pathway have been identified and at least partially characterised, except for one, a postulated enzyme to convert GDP-L-Galactose to L-Galactose-1-Phosphate.
The characterised genes and enzyme activities include the GDP-D-Mannose Pyrophosphorylase (Conklin, 1998; Conklin et al., 1999; Keller et al., 1999), the GDP-D-Mannose 3′,5′-Epimerase (Wolucka et al., 2001; Wolucka and Van Montagu, 2003; Watanabe et al., 2006), the L-Galactose-1-P Phosphatase (Laing et al., 2004; Conklin et al., 2006), L-Galactose Dehydrogenase (Wheeler et al., 1998; Gatzek et al., 2002; Laing et al., 2004), and L-Galactono-1,4-lactone Dehydrogenase (Imai et al., 1998; Bartoli et al., 2005).
The missing enzyme, which (to the best of the applicant's knowledge) has not been reported as being assayed either as an extracted or purified enzyme activity or as an expressed gene, catalyses the second committed step to ascorbic acid biosynthesis.
The VTC2 mutant of Arabidopsis thaliana was identified in a screen for resistance to ozone, and is also characterised as showing especially low ascorbic acid levels (Conklin et al., 2000). The mutated gene was cloned using a map based approach (Jander et al., 2002) and identified as a gene (At4g26850) encoding a novel protein. However this gene was reported to show no homology to other genes in Arabidopsis except for the similarly uncharacterised At5g55120 and other uncharacterised genes from other species. The encoded protein was reported to be most similar to Arabidopsis protein MC015.7, Caenorhabitis elegans protein C10F3.4, and fruitfly (Drosophila melanogaster) protein CG3552, none of which had a demonstrated function.
Although the Arabidopsis gene (At Ag26850) was reported to complement four alleles of the VTC2 mutant, no details were provided (Jander et al., 2002). In addition the authors commented that “although we have a phenotype associated with mutations in VTC2, the regulatory or biosynthetic pathways leading to the reduced vitamin C levels in these mutants remain to be discovered.”
Identification of genes encoding enzymes in the biosynthetic pathway for ascorbate production provides the opportunity for gene-based approaches to manipulation of ascorbate content in plants.
However, although transgenic plants, or mutants, with changed expression of different genes in the L-Galactose pathway have been generated for many of the steps of the L-galactose pathway of ascorbate biosynthesis, and decreased gene expression (and enzyme levels) can result in reduced ascorbate, over expression has not resulted in increased ascorbate in leaves (Ishikawa et al., 2006 and Conklin et al., 2006).
It is an object of the invention to provide improved compositions and methods for modulating GDP-L-Galactose Guanyltransferase (also known as GDP-L-Galactose phosphorylase) activity; and/or GDP-D-Mannose epimerase activity; and/or ascorbate content in plants or at least to provide the public with a useful choice.